Influence of glutamic acid on the endogenous respiration of Bacillus subtilis

Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546-550. 1966.-Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O(2) consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration.     

Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity

Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at > 2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.     

Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water

Some effects of visible light on the survival of Escherichia coli in waters of the Butrón river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using [14C]glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of [14C]glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired [14C]glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.     

Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water.

Some effects of visible light on the survival of Escherichia coli in waters of the Butrón river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using [14C]glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of [14C]glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired [14C]glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.     

ENERGY OF MAINTENANCE IN ESCHERICHIA COLI.

McGrew, Sarah B. (Pennsylvania State University, University Park) and M. F. Mallette. Energy of maintenance in Escherichia coli. J. Bacteriol. 83:844-850. 1962.-Relatively dense populations of Escherichia coli B in log phase were used to detect utilization of exogenous glucose for maintenance without growth. Turbidity at 400 mmu was used as the measure of growth, since it should reflect changes in either cell number or size. A threshold level of glucose was observed below which turbidity did not change during short-time experiments. Repeated additions of glucose during prolonged incubation at 37 C either increased the turbidity slowly or maintained it, depending on the amount of glucose. Plate counts to follow viability showed slow decreases for 10 days, while the unfed controls lost viability quite rapidly. From these results it was concluded that E. coli specifically utilized exogenous glucose for maintenance, without growth. The conflict of this opinion with that of earlier workers is discussed and some implications suggested.     

Starvation-Survival Physiological Studies of a Marine Pseudomonas sp

Starved cultures of a marine Pseudomonas sp. showed a 99.9% decrease in viable cell count during the first 25 days of starvation, yet the culture maintained 10 viable cells per ml for over 1 year. The physiological responses of populations of a marine Pseudomonas sp. to nutrient starvation were observed for periods of up to 40 days. At various intervals during starvation, the numbers of total, viable, and respiring cells were determined within the cultures. The ATP content, endogenous respiration rate, uptake rates, and percent respiration for exogenous glucose and glutamate were determined throughout the starvation period to characterize the physiological changes in the cells. It was observed that, after initial adjustment periods, all parameters tested reached stabilized states after 18 to 25 days of starvation. The results indicate that the actively respiring subpopulation, rather than the viable or total cell numbers, is the most appropriate denominator for interpretation of observed activities on an individual cell basis.     

Synthesis of viral and rRNA in bacteriophage R17 infection of a stringent strain of Escherichia coli.

A previous paper (1973) indicated that infection with bacteriophage R17 permits the synthesis of RNA and spermidine in Escherichia coli (CP78 in the absence of the exogenous essential amino acid, arginine. We have now isolated RNA formed under such conditions and analyzed the newly synthesized species by agarose-acrylamide electrophoresis. It has been shown that infection of the stringent cells in the absence of exogenous arginine resulted in a marked incorporation of uracil into rRNA, as well as into R17 RNA. It was shown that, although the organism was nonauxotrophic for uracil, addition of [-14C]uracil resulted in the rapid formation of TUP, the specific radioactivity of which approached that of the exogenous uracil. This indicated that the incorporation of exogenous uracil into rRNA in R17 infection of the stringent strain reflected a true stimulated synthesis of this nucleic acid. Infection of the essentially isogenic relaxed strain, CP79, under the same conditions inhibited the RNA synthesis to a much less extent than the inhibition caused during the normal infection. These observations provide another example of the close correlation between synthesis of spermidine and of host RNA, even in cells infected by an RNA bacteriophage.     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15°C and 37°C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15 degrees C and 37 degrees C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

Inhibitory effects of H2 on growth of Clostridium cellobioparum.

Hydrogen inhibits the growth of hydrogen-producing Clostridium cellobioparum, but not of Escherichia coli or Bacteroides ruminicola. The inhibition is reversible. When hydrogen was removed either by palladium black or by gassing out the tube, glucose utilization increased as did optical density and hydrogen production of C. cellobioparum. Removal of the H2 by methanogenic bacteria favors the growth of C. cellobioparum. Grown with Methanobacterium ruminantium in various concentrations of glucose, the Clostridium reaches a higher optical density and produces more H2 and a higher viable cell count. The cell yield is also higher than in pure culture. In mixed culture, C. cellobioparum produces more acetic acid and less lactic acid, ethanol, and butyric acid than in pure culture. The significance of this metabolic shift and hydrogen utilization in methanogenesis is discussed.     

Differences in coupling of energy to glycine and phenylalanine transport in aerobically grown Escherichia coli.

Differences exist in the coupling of energy to transport of glycine and phenylalanine in aerobically grown cells of Escherichia coli. Energy derived from respiration, although involved in both uptake systems, is not employed identically as shown by kinetic effects of cyanide and anoxia and by temperature dependencies. Additional evidence for aerobic differences was provided by the effects of azide which greatly decreased initial rates of uptake of glycine but not phenylalanine. The effect on glycine uptake was not due to uncoupling of oxidative phosphorylation or to a decrease in respiration rate. Evidence for anaerobic differences was provided by the addition of either glucose or ferricyanide to cell suspensions containing glycerol, thereby maintaining anoxic uptake of phenylalanine, but not glycine, at the aerobic level. Ferricyanide stimulation required a functional Ca, Mg-adenosine 5'-triphosphatase and involved cell metabolism. Ferricyanide was also found to produce differential stimulation of other amino acid transport systems; tyrosine, tryptophan and leucine uptakes were stimulated whereas those for alanine, proline, threonine, and glutamine were relatively unaffected.     

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.     

Starvation-Survival Physiological Studies of a Marine Pseudomonas sp.

Starved cultures of a marine Pseudomonas sp. showed a 99.9% decrease in viable cell count during the first 25 days of starvation, yet the culture maintained 10⁵ viable cells per ml for over 1 year. The physiological responses of populations of a marine Pseudomonas sp. to nutrient starvation were observed for periods of up to 40 days. At various intervals during starvation, the numbers of total, viable, and respiring cells were determined within the cultures. The ATP content, endogenous respiration rate, uptake rates, and percent respiration for exogenous glucose and glutamate were determined throughout the starvation period to characterize the physiological changes in the cells. It was observed that, after initial adjustment periods, all parameters tested reached stabilized states after 18 to 25 days of starvation. The results indicate that the actively respiring subpopulation, rather than the viable or total cell numbers, is the most appropriate denominator for interpretation of observed activities on an individual cell basis.     

Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.     

Respiration shutoff in Escherichia coli K12 strains is induced by far ultraviolet radiations and by mitomycin C

Ultraviolet radiations (254 nm) (UV) cause respiration to shutoff in Escherichia coli B/r. It has been reported [P.A. Swenson, Photochem. Photobiol., 33 (1981) 855-859 and J. Barbé, A. Vericat and R. Guerrero, Mutation Res., 120 (1983) 1-5] that E. coli K12 strains do not shut off respiration after UV. The latter authors also reported that mitomycin C did not cause this 'SOS' response. In this paper we report that higher UV fluences than were previously used will cause respiration shutoff in K12 strain W3110 and that cyclic AMP increases the sensitivity of respiration shutoff of irradiated cell suspensions. We also report that mitomycin C shuts off respiration in this strain. Neither UV nor mitomycin C causes respiration shutoff in the recA56 derivative of W3110. Thus respiration shutoff is a recA dependent response to UV and mitomycin C in E. coli K12 strains.     

Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down.

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.     

Interaction of silver(I) ions with the respiratory chain of Escherichia coli: an electrochemical and scanning electrochemical microscopy study of the antimicrobial mechanism of micromolar Ag+

Electrochemical techniques were used to study the behavior of Escherichia coli on the addition of 相似文献   

Chlorine injury and the enumeration of waterborne coliform bacteria.

Injury induced in Escherichia coli cells by chlorination was studied from a physiological standpoint. Predictable and reproducible injury was found to occur rapidly in 0.5 mg of chlorine per liter and was reversible under nonselective conditions. There was an extended lag period in the growth of chlorinated cells not seen in control suspensions followed by the resumption of logarithmic growth at a rate equaling that of control cells. The aldolase activity of cells chlorinated in vivo was equivalent to that obtained for control cells. Oxygen uptake experiments showed that chlorinated cells underwent a decrease in respiration that was not immediatedly repaired in the presence of reducing agents. This effect was more pronouned in rich media containing reducing agents. Uptake of metabolities was inhibited by chlorine injury as shown with experiments using 14C-labeled glucose and algal protein hydrolysate.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

The appearance and characterization of cyanide-resistant respiration in the fungus Candida albicans

The respiration of yeast-form cells of the dimorphic fungus Candida albicans became resistant to cyanide during aging treatment in the resting state. An alternative, cyanide-resistant respiratory pathway was found to develop fully in cells aged at a concentration of 0.75 X 10(9)/ml or more at 25 C, but did not appear at 5 C. Chloramphenicol did not prevent the appearance of the alternative respiratory pathway. The effects of inhibitors, salicylhydroxamic acid (SHAM) and disulfiram (tetraethylthiuram disulfide), on respiration of aged cells were examined, and results indicated that SHAM binds at a site on the alternative respiratory pathway whereas disulfiram binds at two sites, one on the conventional respiratory pathway and the other on the alternative pathway. Thus, SHAM is a more selective inhibitor of the alternative respiration of C. albicans cells. SHAM-titration of the alternative respiration revealed that less than 10% of the maximal activity of the alternative respiratory pathway was utilized under normal conditions, indicating that the alternative respiratory pathway makes a small contribution to the total respiration. It was therefore concluded that the alternative, cyanide-resistant respiratory pathway operates fully when the cyanide-sensitive, cytochrome pathway is blocked although aged cells possess both respiratory pathways.